Type 2 diabetes afflicts over 130 million people worldwide, and the incidence is expected to grow steadily over the next several years (Zimmet, P., et al. Nature, (2001) 414, 782). Current treatments are not completely successful in ameliorating Type 2 diabetes for many patients, and more efficacious agents are needed. A major goal of new therapies for Type 2 diabetes is to potentiate the action of insulin.
Insulin signaling is impaired in Type 2 diabetes patients. Although, factors leading to the downregulated insulin signaling pathway is not completely understood, one biochemical event known to disrupt insulin signaling involves the serine phosphorylation of IRS-1 by c-jun N-terminal Kinase 1 (JNK-1). Phosphorylation of IRS-1 at Serine307 greatly reduces the binding affinity of IRS-1 for the insulin receptor (Aguirre, V., et al. J. Biol. Chem. (2000) 275: 9047-9054). This event inhibits IRS-1 tyrosine phosphorylation, and consequently prevents further insulin signal transduction from occuring. A JNK-1 inhibitor could therefore be expected to enhance insulin signaling by preventing the inactivation of IRS-1.
JNK-1 is a member of the mitogen activated protein kinase (MAP kinase) family of enzymes responsible for the serine/threonine phosphorylation of intracellular targets (Kyriakis, J., et al., Physiol. Rev. (2001) 81: 807-869). JNK-1 along with other JNK isoforms, JNK-2 and JNK-3, are activated in response to cellular stresses such as irradiation, hypoxia, chemotoxins, and peroxides. They are also activated in response to various cytokines and participate in the onset of apoptosis. In addition to IRS-1 and IRS-2, other cellular proteins phosphorylated by JNK enzymes include Shc, and Gab-1, and the gene transcription factors Jun, ATF2, Elk-1. JNK-1 and -2 are ubiquitously expressed in human tissues, while JNK-3 is restricted to the brain, heart, and testis, and each isoform is expressed in multiple splice variants. JNK enzymes themselves must be phosphorylated to carry out their functions. The enzymes MKK-4 and MKK-7 are known to phosphorylate and activate JNK-1 and -2.
There is substantial experimental evidence that JNK-1 activity is involved in the pathology of Type 2 diabetes mellitus. Inflammatory cytokines and free fatty acids have been implicated in the development of Type 2 diabetes, and both classes stimulate JNK activation (Chang, L. et al., Nature (2001) 410: 37-40). Insulin gene expression is reduced, while JNK is activated under conditions of oxidative stress. Furthermore, suppression of the JNK pathway protects pancreatic β-cells from oxidative stress (Kaneto, H., et al., J Biol Chem (2002) 277:30010-8). JNK may also mediate endothelial cell apoptosis caused by diabetes-associated hyperglycemia, and pancreatic β-cell apoptosis associated with diabetes (Ho, F. M. et al., Circulation (2000) 101:2618-2624). Activation of JNK is involved in tumor necrosis factor (TNF) induced lipolysis in adipocytes. Human genetic evidence shows that increased JNK activity caused by loss-of-function mutations in the JNK scaffold protein JIP1 is causal to Type 2 diabetes (Waeber G. et al. Nature Genet. (2000) 24: 291-295). JNK-1 is also involved in the mechanism of hepatic insulin resistance caused by liver steatosis (Samuel V T, et al. J. Biol. Chem. 2004, 279:32345-32353).
The most compelling data supporting an integral role for JNK-1 in insulin action and obesity comes from the targeted disruption of the JNK-1 gene in mice (Hirosumi J, Nature (2002) 420:333-336). Absence of JNK-1 protects mice from diet-induced obesity, and results in decreased adiposity and enhanced secretion of adiponectin. The JNK-1 null mice maintain lower fasting plasma glucose and insulin levels compared to their wild type littermates when they are high fat fed, indicating that these animals are protected from the development of obesity-induced insulin resistance. They also demonstrate greater insulin sensitivity in both oral glucose and intraperitoneal insulin tolerance tests. In addition, heterozygous animals show a partial phenotype. Moreover, genetically obese mice (ob/ob) with targeted mutations in JNK-1 were leaner and maintained lower blood glucose and insulin levels compared with the ob/ob mice expressing fuctional JNK-1. JNK-1 phosphorylation of IRS-1 at Ser307 has been shown to downregulate insulin signaling in vitro. The extent of IRS-1 Ser307 phosphorylation is markedly decreased in obese JNK-1 null mice, while the insulin-induced IRS-1 tyrosine phosphorylation is strongly enhanced in livers of those mice. Interestingly, ablation of the JNK-1 gene generates a phenotype of increased insulin sensitivity that is not mimicked by deficiency of JNK-2.
There is potential for JNK inhibitors to treat human diseases besides Type 2 diabetes. JNK-2 null mice have been produced by two different laboratories, and there is a clear role for JNK-2 in immunological function (Yang, D. D., et al., Immunity, (1998) 9: 575-585; Sabapathy, L. et al., Curr. Biol. (1999) 9:116-125). JNK-2, often in concert with JNK-1, has been implicated in the pathology of autoimmune disorders such as rheumatoid arthritis (Han, Z., et al., Arthritis & Rheumatism (2002) 46: 818-823), and asthma. It also may play a role in cancer (Zhang, H., et al., J. Biol. Chem. (2002) 277: 43648-43658), as well as ischemia-reperfusion injury following myocardial infarction (Kumar, D., et al., Mol. Cell. Biochem. (2004) 258: 211-218) or stroke. Inhibitors of JNK-1 and JNK-2 could be useful for treating these diseases, as well as a broad range of other diseases with an inflammatory component.
Human JNK-3 expression is limited to the brain, heart, and testis. JNK-3 has been shown to mediate neuronal apoptosis and may therefore be involved in the pathology of neurodegenerative diseases. Inhibitors of JNK-3 could be useful for treating Parkinson's disease, Alzheimer's disease, epilepsy, stroke, and other diseases of the central nervous system as JNK3 knockout mouse studies suggest the role of JNK-3 in neurological disorders (Kuan, C-Y., et al., PNAS, USA. (2003) 100:15184-15189).